Mechanisms responsible for epithelial HCO3-transport are of great importance to renal physiology. The renal tubule reabsorbs filtered HCO3 and generates "new HCO3" in order to maintain systemic acid-base homeostasis. The HCO3-transporters NBCe1& AE1, and carbonic anhydrases CA II & CAIV are proteins that play important roles in HCO3-reabsorption by the renal tubule. Recent studies on AE1, and preliminary data on NBCel, suggest that these HCO3-transporters bind to both CA II and CA IV and form HCO3- transport metabolons. The specific aims of this project are to use X-ray crystallography to describe the structural basis of 1) the intracellular interaction between CA II and the HCO3-transporters, and 2) the extracellular interaction between CA IV and the HCO3-transporters. The approach will be to generate recombinant peptides that correspond to the CA-II and CA-IV binding domains of AE1 and NBCe1 and cocrystallize them with their respective CA. In addition, binding properties of the protein-protein interactions will be characterized using such techniques as solid-phase binding and pull-down assays. Understanding the interactions between CAs and HCO3-transporters will reveal important features of how these proteins interact with one another and may reveal how their interactions influence the function of AE1 and NBCe1.The proposed study will provide key details on the finer mechanisms of renal HCO3 transport, which will provide a better understanding of the physiology and importance of the studied proteins to kidney function.